Male C57Bl/6 mice were obtained from Charles River (Sulzfeld, Germany) and acclimatized for a week prior to the experiment. At 6 wk of age, they were randomized into two groups equal in weight: High-fat (n = 26; 60E% fat, D12492; Research Diets, New Brunswick, NJ, United States) and control (n = 24; 10E% fat, D12450B; Research Diets). Diets were paired for fiber and protein content. Mice were housed six per cage in a standard animal laboratory with a dark/light cycle of 12/12 h, with free access to food and water. After 15 wk of feeding, mice were euthanized with a gas mixture of CO₂ (70%) and O₂ (30%) (AGA, Riihimäki, Finland), and cervical dislocation. Animal experiments were approved by the National Animal Experiment Board of Finland.

Fresh segments of duodenum, jejunum, ileum and proximal colon were collected in duplicate, opened along the mesenteric border, pinned onto 0.3 cm² sliders and mounted into an EasyMount Ussing chamber system with a voltage-clamp apparatus (Physiologic Instruments, San Diego, CA, United States). Two intestinal segments were collected in duplicate from each mouse, because only four chambers were available. Tissues were surrounded by 5 mL Ringer solution (120 mmol/L NaCl, 5 mmol/L KCl, 25 mmol/L NaHCO₃, 1.8 mmol/L Na₂HPO₄, 0.2 mmol/L NaH₂PO₄, 1.25 mmol/L CaCl₂, 1 mmol/L MgSO₄, 10 mmol/L glucose) on each side. The system was water-jacketed to 37 °C and carbonated with a carbogen (95% O_{2 ,} 5% CO₂; AGA) gas flow. After an equilibration period of 10 min, solutions were replaced with fresh Ringer, and 4 kDa FITC-dextran (TdB Cons, Uppsala, Sweden) was added to the luminal side to a final concentration of 2.2 mg/mL. Resistance and short-circuit current were followed for 60 min, after which, serosal fluorescence was detected with a Wallac Victor² 1420 Multilabel counter (Perkin-Elmer, Waltham, MA, United States). Intestinal permeability was determined by comparing serosal fluorescence to luminal fluorescence as per mille of translocated dextran.

Feces were collected at 13 wk by placing mice individually in metabolic cages for 24 h. Feed and water was provided ad libitum. Feces were carefully separated from all other material and frozen at -20 °C. Upon analysis, feces of six mice from each group were dried overnight with nitrogen and pulverized. Bile acids were extracted and measured by gas-liquid chromatography according to the method by Grundy et al[22] in the laboratory of the Hospital District of Helsinki and Uusimaa. Internal standards were run for isolithocholic acid, lithocholic acid, epideoxycholic acid, DCA, chenodeoxycholic acid, cholic acid and UDCA.

Segments of jejunum were collected from each mouse, snap frozen in liquid nitrogen, and stored at -80 °C. RNA was extracted with TRI Reagent (RT111; Molecular Research Center, Cincinnati, OH, United States) according to the manufacturer’s protocol. Tissues were homogenized with Precellus24 (6500 rpm, 2 × 15 s; Bertin Technologies, Montigny-le-Bretonneux, France). Phase separation was performed with chloroform (34 854; Sigma-Aldrich, St Louis, MO, United States), and RNA precipitation with isopropanol (P/7507/15X, Fisher Scientific, United States). RNA concentration was measured with NanoDrop 8000 (Thermo Scientific, Waltham, MA, United States), and converted to cDNA with a SuperScript VILO cDNA synthesis kit (Applied Biosystems, Carlsbad, CA, United States) according to the manufacturer’s instructions, using 1 μg RNA in a reaction volume of 20 μL. Reactions for quantitative real time polymerase chain reaction (qPCR) were run using TaqMan chemistry (Applied Biosystems) for FXR (Mm00436420_m1) and tumor necrosis factor (TNF) (Mm00443258_m1). Gene expression was normalized to beta-actin and beta-glucuronidase. Reactions were run on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, United States) in triplicate. Skeletal muscle was used as a non-expressing negative control. Gene expression was calculated with Bio-Rad CFX Manager software using the normalized expression ΔΔC(t) method.

Permeability results were statistically analyzed with a Mann-Whitney U test due to the uneven distribution of values around the means. In addition to calculating intestinal permeability for each segment (duodenum, jejunum, ileum and colon), a value for overall intestinal permeability was calculated for each mouse as an average of the permeability in its two measured intestinal segments. Bile acid and gene expression data were analyzed with a t test between control and high-fat groups. As there was a special focus on UDCA, the concentration of this bile acid relative to total identified bile acids was also calculated. The gene expression data had a setting of four groups, characterized by two factors possibly affecting intestinal FXR and TNF expression: intestinal segment and diet. The independent effects of these two factors were statistically analyzed with a factorial experiment. Groups were also compared with each other individually for both intestinal segments with a t test. Equality of variances was tested with Levene’s test. All statistical analyses were performed with PASW Statistics version 18.0.2 (IBM, United States). All data are expressed as mean ± SE unless otherwise stated.

The weight of the high-fat-fed mice was significantly higher compared to control mice (49.5 ± 0.59 g vs 28.6 ± 0.36 g, P < 0.001). High-fat-feeding increased intestinal permeability significantly in the jejunum (median 0.334 per mille translocated dextran for control vs 0.393 for high-fat, P = 0.03) and colon (median 0.335 for control vs 0.433 for high-fat, P = 0.01), but not in the duodenum (median 0.359 for control vs 0.360 for high-fat, P = 0.33) or ileum (median 0.351 for control vs 0.452 for high-fat, P = 0.69, Figure 1). Resistance and short-circuit current were measured during the Ussing chamber experiments to monitor tissue viability. The stability of these values indicates good viability of tissue segments throughout the experiments (Figure 2).

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Figure 1 Effect of high-fat-feeding on intestinal permeability. Intestinal permeability in duodenum (A), jejunum (B), ileum (C), and colon (D) of high-fat-fed vs control mice. Permeability was measured in an Ussing chamber. Results are shown as per mille translocated dextran. Box plots show median, upper and lower quartiles, and Tukey’s whiskers (highest and lowest values, outliers shown as black dots). ^aP < 0.05 between high-fat and control.

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Figure 2 Stability of Ussing chamber experiments. Short-circuit current (A) and resistance (B) in Ussing chamber experiments. Bars indicate mean and SEM. n = 9-15/group.

Feces from high-fat fed mice contained substantially more bile acids compared to feces from control mice (2.13 ± 0.29 mg/g dry feces vs 0.37 ± 0.03 mg/g dry feces, P < 0.001; Figure 3A). This was reflected as a significantly elevated concentration of each bile acid (P < 0.01) except isolithocholic acid (Figure 3B). We were especially interested in the effects of UDCA, which is considered cytoprotective, therefore, we calculated the ratio of UDCA to total bile acids. The proportion of UDCA in feces of high-fat-fed mice was nearly halved compared to that in control mice (1.4% ± 0.1% in high-fat vs 2.8% ± 0.3% in controls, P < 0.01, Figure 3C). There was also a marked inverse correlation of overall intestinal permeability to the proportion of UDCA in total fecal bile acids (r = -0.72, P = 0.01, n = 11, Figure 4A). The correlation remained significant even after controlling for body weight (partial correlation r = -0.64, P < 0.05, n = 11). A trend for a similar association was seen in the subgroups of jejunal and colonic permeability (r = -0.88, P = 0.05, n = 5 for jejunum and r = -0.70, P = 0.12, n = 6 for colon, Figure 4B and C). In addition, we correlated permeability to the ratio of UDCA to DCA, a cytotoxic bile acid. This was done to prevent bias by the unmeasured muricholic acid, which is a primary bile acid present in murine bile[23], and is left unidentified by methods designed for clinical use. The relation of UDCA to DCA showed a similar halved concentration and inverse correlation to permeability (r = -0.70, P = 0.02).

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Figure 3 Fecal bile acids. A: Concentration of total measured bile acids; B: Concentration of measured bile acids in feces; C: Proportion of ursodeoxycholic acid (UDCA) in total measured bile acids. Bars indicate mean and SEM. n = 6/group, ^aP < 0.001, ^bP < 0.01 between high-fat and control groups.

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Figure 4 Correlation of proportion of ursodeoxycholic acid with intestinal permeability. Pearson’s correlation of fecal proportion of ursodeoxycholic acid (UDCA) percentage to overall permeability of intestine (A), jejunal permeability (B), and colonic permeability (C). Each dot represents an individual animal.

The effect of high-fat-feeding on bile acid metabolism and intestinal inflammation was assayed as expression of intestinal FXR and TNF in the jejunum and colon. Jejunal FXR expression was increased 30% by high-fat feeding (0.74 ± 0.049 for high-fat vs 0.57 ± 0.051 for control, P = 0.03) and colonic TNF expression was doubled (0.82 ± 0.148 for high-fat vs 0.42 ± 0.068 for control, P = 0.02) but no significant differences were seen in jejunal tumor necrosis factor (TNF) (P = 0.30) or colonic FXR (P = 0.63, Table 1). FXR and TNF expressions did, however, correlate with each other (r = 0.41, P < 0.01, n = 50, Figure 5).

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Figure 5 Correlation of tumor necrosis factor and farnesoid X receptor expression. Pearson’s correlation of tumor necrosis factor (TNF) and farnesoid X receptor (FXR) expression. Jejunal values are shown as black diamonds and colonic values as grey squares. Each dot represents an individual animal.

A factorial experiment on the independent effects of diet and intestinal segment on gene expression revealed that overall intestinal TNF expression was elevated by high-fat-feeding (P = 0.02). Moreover, both TNF and FXR expressions were higher in colon compared to jejunum (P < 0.001 for both).

Intestinal permeability has recently been linked to type 2 diabetes, steatosis and steatohepatitis. One proposed cause for increased permeability is a diet high in fat. A high-fat-diet increases excretion of bile. Secondary bile acids are known to increase permeability of epithelial monolayers in vitro. However, physiological concentrations of bile juice obtained from healthy rats have failed to increase epithelial monolayer permeability. This may be due to the fact that bile consists of a profile of several bile acids, which differ in cytotoxicity. It has not yet been addressed whether high-fat feeding alters the profile of fecal bile acids, and whether this profile plays a role in the onset of barrier dysfunction.

Proposed mechanisms for the dietary induction of increased intestinal permeability have mostly been related to alterations in gut microbiota. Only a few publications have mentioned other luminal factors affecting permeability. The research hotspot in this field is how diet modifies bile acid metabolism and leads to increased intestinal permeability.

Although bile is considered detrimental to the gut epithelium, physiological concentrations have not increased epithelial monolayer permeability. We show that not only was bile excretion increased by high-fat feeding, but the profile of fecal bile acids had become more cytotoxic than in healthy control mice. The proportion of a hydrophilic bile acid, ursodeoxycholic acid (UDCA), was decreased by high-fat feeding and correlated inversely with intestinal permeability.

The prevention of barrier dysfunction may decrease the risk of its associated diseases. The present results suggest that bile acid metabolism is a potential target for the prevention of a barrier dysfunction.

Intestinal permeability refers to how large molecules, such as inflammatory bacterial components, translocate through the intestinal epithelium into the circulation. Translocation may be paracellular, through tight-junction proteins, or transcellular, via chylomicrons. UDCA is a hydrophilic bile acid. Deoxycholic acid is a secondary bile acid produced from cholic acid by intestinal microbes.

The authors tested intestinal permeability in an ex vivo model (Ussing chambers; mucosal to serosal flux of FITC-labeled dextran) in mice on a high-fat diet; moreover, analysis of fecal bile acids and expression of mucosal tumor necrosis factor and farnesoid X receptor was performed.